U.S. patent application number 09/808408 was filed with the patent office on 2002-03-21 for agents and methods for the prevention of initial onset and recurrence of existing cancers.
This patent application is currently assigned to Oncology Sciences Corporation. Invention is credited to Alworth, William, Kumar, Addanki P., Slaga, Thomas J..
Application Number | 20020035098 09/808408 |
Document ID | / |
Family ID | 25198677 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020035098 |
Kind Code |
A1 |
Slaga, Thomas J. ; et
al. |
March 21, 2002 |
Agents and methods for the prevention of initial onset and
recurrence of existing cancers
Abstract
The use of 2-methoxyestradiol, analogues of 2-methoxyestradiol,
their method of synthesis and therapeutic use, and the use of
combinations of the 2 methoxyestradiol and its analogues with
synergistic compounds (namely eugenol), all in the prevention of
initial onset cancers and the recurrence of previously existing
cancers.
Inventors: |
Slaga, Thomas J.; (Denver,
CO) ; Kumar, Addanki P.; (Denver, CO) ;
Alworth, William; (New Orleans, LA) |
Correspondence
Address: |
DAVID G. HENRY
900 Washington Avenue
P.O. Box 1470
Waco
TX
76701
US
|
Assignee: |
Oncology Sciences
Corporation
Austin
TX
|
Family ID: |
25198677 |
Appl. No.: |
09/808408 |
Filed: |
March 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09808408 |
Mar 14, 2001 |
|
|
|
09527283 |
Mar 17, 2000 |
|
|
|
Current U.S.
Class: |
514/169 |
Current CPC
Class: |
A61K 31/58 20130101;
A61K 31/567 20130101; A61K 31/085 20130101; A61K 31/566 20130101;
A61P 35/00 20180101; A61K 31/565 20130101; A61K 31/567 20130101;
A61K 31/565 20130101; A61K 31/085 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/169 |
International
Class: |
A61K 031/56 |
Claims
We claim:
1. The use of compositions useful in the inhibition of cancerous
cell proliferation selected from a group consisting of: the
2-ethyl-17-.beta.-estradiol molecules identified as analogues 20-22
in FIG. 3, specifically excluding any claim to
2-methyloxyestradiol; the 17-.alpha.-ethynyl molecules identified
as analogues 23-26 in FIG. 3; the 17-.alpha.-ethyl molecules
identified as analogues 27-30 in FIG. 3; the 2,3-methylenedioxy
molecules identified as analogues 31, 32, and 33 in FIG. 4; the
2-alkoxy substituted analogues of estrone molecules identified as
analogues 8-10 in FIG. 1; the 2-ethyl substituted molecule
identified as analogue 14 in FIG. 1; and the
2,3-methylenedioxyestrone molecule identified as analogue 18 in
FIG. 2.
2. A method for inhibiting cancerous cell proliferation comprising
the steps of: selecting a composition from the group consisting of
the 2-ethyl-17-.beta.-estradiol molecules identified as analogues
20-22 in FIG. 3, specifically excluding any claim to
2-methyloxyestradiol, the 17-.alpha.-ethynyl molecules identified
as analogues 23-26 in FIG. 3, the 17-.alpha.-ethyl molecules
identified as analogues 27-30 in FIG. 3, the 2,3-methylenedioxy
molecules identified as analogues 31, 32, and 33 in FIG. 4, the
2-alkoxy substituted analogues of estrone molecules identified as
analogues 8-10 in FIG. 1, the 2-ethyl substituted molecule
identified as analogue 14 in FIG. 1, or the
2,3-methylenedioxyestrone molecule identified as analogue 18 in
FIG. 2; and administering said composition to cells in which is
identified suspected cancer cells.
3. The method of claim 2 wherein said suspected cancer cells are
brain cancer cells.
4. The method of claim 2 wherein said suspected cancer cells are
nervous system cancer cells.
5. The method of claim 2 wherein said suspected cancer cells are
brain cancer cells and nervous system cancer cells.
6. The method of claim 2 wherein said suspected cancer cells are
prostate cancer cells.
7. A composition for application to cancerous cells consisting in
active constituents substantially of one or more agents chosen from
the 2-ethyl-17-.beta.-estradiol molecules identified as analogues
20-22 in FIG. 3, specifically excluding any claim to
2-methyloxyestradiol, the 17-.alpha.-ethynyl molecules identified
as analogues 23-26 in FIG. 3, the 17-.alpha.-ethyl molecules
identified as analogues 27-30 in FIG. 3, the 2,3-methylenedioxy
molecules identified as analogues 31, 32, and 33 in FIG. 4, the
2-alkoxy substituted analogues of estrone molecules identified as
analogues 8-10 in FIG. 1, the 2-ethyl substituted molecule
identified as analogue 14 in FIG. 1, or the
2,3-methylenedioxyestrone molecule identified as analogue 18 in
FIG. 2.
8. The method of claim 8 wherein said suspected cancer cells are
brain cancer cells.
10. The method of claim 8 wherein said suspected cancer cells are
nervous system cancer cells.
11. The method of claim 8 wherein said suspected cancer cells are
brain cancer cells and nervous system cancer cells.
12. The method of claim 8 wherein said suspected cancer cells are
prostate cancer cells.
13. A method for preventing the onset of cancer and for preventing
the recurrence of cancer comprising the administration of a
therapeutic dose to a human recipient of one or more compositions
selected from the group consisting of: 2-methoxyestradiol;; the
2-ethyl-17-.beta.-estradiol molecules identified as analogues 20-22
in FIG. 3, specifically excluding any claim to
2-methyloxyestradiol; the 17-.alpha.-ethynyl molecules identified
as analogues 23-26 in FIG. 3; the 17-.alpha.-ethyl molecules
identified as analogues 27-30 in FIG. 3; the 2,3-methylenedioxy
molecules identified as analogues 31, 32, and 33 in FIG. 4; the
2-alkoxy substituted analogues of estrone molecules identified as
analogues 8-10 in FIG. 1; the 2-ethyl substituted molecule
identified as analogue 14 in FIG. 1; and the
2,3-methylenedioxyestrone molecule identified as analogue 18 in
FIG. 2.
14. The method of claim 13 wherein a therapuetic dose of eugenol is
administered in conjunction with said one or more compositions.
15. The method of claim 13 wherein said cancer is human prostate
cancer.
16. The method of claim 14 wherein said cancer is human prostate
cancer.
Description
CITATION TO PRIOR APPLICATION
[0001] This is a continuation-in-part with respect to U.S.
application, Ser. No. 09/527283, filed Mar. 17, 2000 from which
priority is claimed under 35 U.S.C. .sctn.120 and under provisions
of the Patent Cooperation Treaty.
BACKGROUND OF THE INVENTION
[0002] A. Field of the Invention
[0003] The present invention concerns the novel use of known
chemical compounds, the chemical synthesis and use of novel
chemical compounds, and the use of novel combinations of both new
and existing compounds, all in the prevention of initial onset and
recurrence of a broad array of cancers.
[0004] B. Background of the Invention
[0005] 1. The Problem: Primary Modalities of Cancer Cell Growth and
Expansion (New. Existing and Recurring Cancers)
[0006] Cancer is the second leading cause of death in the United
States, accounting for approximately one in four deaths. Recent
estimates by the American Cancer Society suggest that in excess of
500,000 people die from cancer every year--that is approximately
1,500 deaths a day. Further, approximately 2.5 million new cases of
cancer were expected to be diagnosed in the year 2000 alone. At an
estimated annual cost of $107 billion dollars in health care costs
and lost productivity due to death and illness, cancer inflicts a
vast human and monetary toll on the United States.
[0007] The generic use of the term "cancer" only hints at the vast
diversity of anatomical structures that this disease affects and
the myriad of molecular bases that form the foundation of this
disease. The collective use of the word cancer includes diseases
affecting the brain, breast, cervix uteri, colon, corpus uteri,
kidney, renal pelvis, larynx, lung, bone marrow, bronchus, skin,
lymph system, nervous system, oral cavity, pharynx, ovary,
pancreas, prostate, rectum, stomach, testis, thyroid, urinary
bladder, and others. The individual molecular bases of these
diverse afflictions can be varied and diverse. However, among this
diverse field of afflictions, there exist two unified modalities of
cell growth and/or proliferation that are common to almost all
types of cancer: 1) unchecked cell growth and/or immortality, and
2) angiogenesis.
[0008] On of the problems that characterize a vast number of
cancers is the unregulated growth or unchecked life span of
aberrant cells in the various tissues of the body. Normal cells
grow, divide, and die on a regular basis. The process by which
cells normally die is called apoptosis. However, when normal cell
growth and death become unchecked in the body, by any number of
processes, such unchecked growth and/or immortality leads to
destroy the regular functioning of the various tissues of the body.
Such growth or immortality can ultimately lead to the occurrence of
a host of solid tumors, leukemia's, lymphomas, or the metastasis of
cancer cells throughout the body. Unchecked cell growth and/or
immortality are problematic biological mechanisms common to almost
all types of cancer.
[0009] Another biological mechanism that is common to, and
problematic in the prevention or treatment of, all solid cancer
tumors is angiogenesis. Angiogenesis refers to the process by which
new blood vessels are formed in the body. Without a dedicated blood
supply, solid tumors have only limited growth potential--perhaps 2
mm in diameter maximum. However, angiogenesis often occurs in
cancerous tissues and tumors, thus enabling solid tumors to
sequester greater amounts of nutrients from the body and allowing
them to proliferate rapidly, even spreading to other parts of the
body. Angiogenesis is a critical means by which solid tumors grow
rapidly and metastasize, hastening the process of death or
disfigurement.
[0010] These two independent biological mechanisms are the common,
primary modalities by which almost all cancer cells proliferate and
grow. Hence, a novel approach for the treatment of cancer would be
the development of pharmacological agents that have dual roles as
anti-angiogenic as well as pro-apoptotic agents. Such an agent will
have the ability to target both components of a cancer: kill the
tumor cell by induction of apoptosis and cut off the blood supply
to the tumor cell so that it will not grow.
[0011] In addition to the dire need for effective treatment
modalities for existing cancers, there is arguably an even greater
need for effective cancer preventative means, both with respect to
initial onset of cancers, as well as in the context of recurrence
of cancers after operative intervention.
[0012] A recent breakthrough in the treatment of cancer is the use
of 2-methoxyoestradiol (hereinafter "2-ME"). 2-ME is an endogenous
non-toxic metabolic byproduct of estrogens that is present in human
urine and blood. (1) A potential role for 2-ME as a chemopreventive
agent has been reported in the mammary and pancreatic models. (2)
2-ME has also been shown to inhibit endothelial cell proliferation
implicating its potential role in angiogenesis. (3) In addition,
apoptosis has been implicated as a mechanism for 2-ME's cytostatic
and anti-angiogenic effect.
SUMMARY OF THE INVENTION
[0013] It is an object of the present invention to provide an agent
or composition, or more than one agent or composition, that is
efficacious in inhibiting the proliferation and/or angiogenesis of
cancer cells.
[0014] It is another object of the present invention to provide a
method for creating novel molecules that are efficacious in
inhibiting the proliferation and/or angiogenesis of cancer
cells.
[0015] It is another object of the present invention to provide a
composition the primary active ingredient of which are an analogue
or analogues of 2-methoxyestradiol which are efficacious in
inhibiting the proliferation and/or angiogenesis of cancer
cells.
[0016] It is another object of the present invention to provide a
method for inhibiting the proliferation and/or angiogenesis of
cancer cells through use of a composition the primary active
ingredient of which is 2-methoxyestradiol or an analogue thereof,
as described herein.
[0017] It is, therefore, an object of the present invention to
provide a new modality for the prevention of cancers as well as the
suppression of recurrence of cancers once treated.
[0018] It is another object of the present invention to provide a
new modality for the prevention of cancer as well as the
suppression of recurrence of cancers once treated.
[0019] It is another object of the present invention to provide a
new modality for the prevention of prostate cancer as well as the
suppression of recurrence of prostate cancer once treated.
[0020] It is another object of the present invention to provide a
method by which the known substance of 2-ME may be employed in a
new and unobvious manner in the prevention of initial onset and of
recurrence of cancers, including prostate cancer.
[0021] It is another object of the present invention to provide a
method by which the known substance of 2-ME, alone, or in
combination with synergistic compounds, including eugenol and
certain other herein disclosed compounds, may be employed in the
prevention of initial onset and post-operative recurrence of
cancers, including prostate cancer.
[0022] In satisfaction of these and related objects, disclosed and
claimed herein is the use of 2-ME or 2ME analogues, alone, or in
combination with synergistic compounds, including eugenol and
certain other herein disclosed compounds, in the prevention of
initial onset and post-operative recurrence of cancers, including
prostate cancer. Also, because of the mode of action
(anti-tumorigenic, anti-angiogenic, and pro-apoptotic), 2-ME or
certain herein disclosed derivatives, with or without the herein
described synergistic compounds, can be used, not only in the
treatment of any kind of tumor, including prostate, brain, liver,
lung, colon, and skin, but in preventing initial onset, or
recurrence of such cancers after treatment.
[0023] Findings by the present inventors pertaining to the
mechanisms of action of 2-ME, its derivatives, and the synergistic
compounds herein described indicate that these compounds serve as
cancer preventative agents, as well as curative agents. The present
inventors previous work, filed with the original patent application
and another continuation-in-part application, shows that 2-ME is of
great significance in the treatment of prostate, brain, and nervous
system cancer through the induction of apoptosis. This body of work
indicates that 2-ME is an anti-tumorigenic agent with a significant
therapeutic advantage since it can preferentially inhibit actively
proliferating cells (characteristic of tumor cells) without
affecting the growth of normal cycling cells. Additionally, 2-ME
appears to also inhibit the formation of new blood vessels. To the
best of our knowledge, this is the first compound that targets two
components of cancer: the tumor cells and their blood supply. The
present inventors have demonstrated that 2-ME is a chemical
compound with a significant role as an antitumorigenic agent with
broad efficacy in a variety of cancerous cell populations.
[0024] Building on these findings, further experiments have helped
to elucidate the structural bases for 2-ME's molecular efficacy. A
number of experiments have been conducted using 2-ME and
16-epiestriol (hereinafter "16-ES"), an analogue of 2-ME that lacks
the methoxy group at the second position. In a multitude of
experiments, using prostate cancer cell lines (both
androgen-dependent (LNCaP), and androgen-independent (DU145)
cells), and a brain and/or nervous system cancer cell line (DAOY),
the present inventors have studied the effects of 2-ME and 16-ES on
cell proliferation and the induction of apoptosis, in a number of
ways. The sum of all the data clearly indicates that 2-ME is a
compound that significantly inhibits cancerous cell growth and has
pro-apoptotic effects, while 16-ES does not. In total, these data
show that the efficacy of 2-ME is associated with the methoxy
moiety at the second position of 17.beta.-estradiol (E.sub.2).
Further, it also indicates efficacy of a series of compounds with
various moieties at the second position in the treatment of cancer.
Additionally, the specific anti-proliferative, pro-apoptotic,
anti-angiogenesis, and other efficacy of 2-ME against cancer cells
indicates that other structural modifications of the molecule will
reasonably be expected to increase the efficacy of the agent. Thus,
the present inventors now propose a method of synthesizing a number
of analogues of 2-ME that will exceed the efficacy of 2-ME in the
prevention of cancer.
[0025] Further still, the investigations of the present inventors
indicate that 2-ME (and predictably its analogs) work
synergistically with other compounds, notably eugenol, to achieve
even greater results in the same manner and modality as 2-ME alone
in attacking cancer cells and preventing initial cancer formation
and recurrence of cancer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0026] Data from the present inventors laboratory shows that 2-ME
inhibits the growth of brain, nervous system and prostate cancer
cells but that 16-epiestriol does not. This indicates that
substituting the second position of 17b-estradiol (E.sub.2) with a
methoxy group generates a molecular structure that shows
significant and selective growth inhibitory activity toward
prostate cancer cells while simultaneously eliminating the
potentially detrimental growth stimulating activity of E.sub.2
itself. The analogues of 2-ME to be prepared as described below are
designed (1) to determine which components of the 2-ME molecule in
addition to the 2-methoxy group are required for the observed
chemopreventive effects and (2) to determine if growth-inhibitory
2-ME analogues can be created that are effective.
[0027] The initial compounds to be synthesized will be 2 alkoxy
substituted analogues of estrone shown in FIG. 1. These compounds
will then be converted into the 2-ME analogues as shown in FIG. 3
(analogues 19-21, 23-25, and 27-29).
[0028] FIG. 1 illustrates how the A ring of the E.sub.2 steroidal
nucleus will be modified to generate 2-alkoxy substituted analogues
of estrone (analogues 8-10) and a 2-ethyl substituted estrone
analogue (analogue 14). The key reactions in this figure are the
synthesis of compound 2,2,4-diiodoestrone, and its conversion to
compound 3, the 2-iodoestrone derivative. The iodination and
diodination of the estrone starting material (analogue 1) will be
carried out as described by Ikegawa et al in their synthesis of
catecholic equilin and equilin derivatives. (4) The proposed
conversion of the ethylenedioxy protected 2-iodoestrone derivative
4 to the protected 2-methoxy, 2-ethoxy, and 2 benzyloxy derivatives
5-7 by Cu (I) catalyzed reactions of the alkoxides in
dimethylformamide in the presence of a crown ether is based upon
the comparable reaction of a protected 2-iodoequilin also described
by Ikegawa et. al in the synthesis of catechol equilins. (4) It
should be noted that if it proves necessary the estrone starting
material used in FIG. 1 could be protected as the ethylenedioxy
derivative by treatment with ethylene glycol prior to the
iodination reaction. The Pd(Ph.sub.3)Cl.sub.2/CuI catalyzed
coupling of the aryl iodide (analogue 4) with trimethylsilyl
substituted acetylene to yield the 2-alkynyl substituted estrone
derivative 11 shown in FIG. 1 has many known precedents (5). The
present inventors have carried out many such coupling reactions in
their laboratory and have found that molecules containing active
hydrogens (NH.sub.2 or OH groups) can be successfully coupled in
such reactions if care is taken to form the reactive Cu-TMS
acetylene complex before the halogenated aromatic substrate is
added. It is therefore anticipated that this reaction will proceed
as shown in FIG. 1. If, however, the reaction fails to be
successful as shown in FIG. 1, the intermediate 4 will be coupled
with trimethylsilylacetylene in 9:1 CH.sub.3CN/H.sub.2O catalyzed
with Pd(AcO).sub.2/PPh.sub.3/CuI. The present inventors have
carried out a model reaction in their laboratory with an
unprotected iodophenol that gave the desired coupling product with
this procedure.
[0029] FIG. 2 outlines the reaction sequence that will be employed
to prepare the 2,3-methylenedioxyestrone derivative (analogue 18).
This reaction sequence is based upon the reaction sequence employed
by Stubenrauch and Knuppen to prepare catechol estrogens. (6)
[0030] FIGS. 3 and 4 illustrate how 2-methoxyestrone and the
2-methoxyestrone analogues prepared as outlined in FIGS. 1 and 2
above will be converted into (i) 2-methoxyestrone and its analogues
and (ii) 2,3-methylenedioxyestrone analogues modified at position
C-17. The preparation of these structures will not only allow us to
test the requirement for the 17b-hydroxyl group in the
chemopreventive activity of 2-ME but will also enable us to
determine if substitutions at C-17 (for example, the 17-ethynyl2-ME
derivative, 23) will decrease the rate of metabolism and
deactivation of 2-ME and its analogues. As outlined in FIGS. 3 and
4 below, the present inventors propose to prepare both
2-ethyl-17b-estradiol (analogue 22) and
2,3-methylenedioxy-17b-estradiol (analogue 32). In addition, since
17a-ethynylestradiol (ethynylestradiol) is both a potent estrogenic
and long-lived analogue of E.sub.2, the 17a-ethynyl derivative of
2-ME (analogue 19) will be prepared as outlined in FIG. 3. In
addition, by directing synthesis to produce estrone analogues of
the target structures (analogues 8-10, 14, and 18) as illustrated
in FIGS. 1 and 2, it will be possible to prepare 17a-ethynyl, and
17a-ethyl derivatives of the 2-alkoxy, 2-ethyl, and
2,3-methylenedioxy analogues (analogues 23-26, 27-30, 31 and
32).
[0031] It should be noted that the proposed reactions used to
modify the C-17 carbonyl of the estrone analogues shown in FIGS. 3
and 4 are standard reactions that have been successfully applied to
estrone. (7)
[0032] Although not explicitly shown in FIGS. 1 and 3, the
2-ethynyl intermediate shown in FIG. 1 (analogue 12) will also be
converted into 2-ethynylestrone and 2-ethynylestradiol for testing.
Further, although not explicitly indicated in FIGS. 1 and 2, the
2-ethynylestrone derivative 11 shown in FIG. 1 will also be
converted into 2-ethynylestrone and 2-ethynylestradiol as shown in
FIG. 2 for the other intermediates. This will generate two
additional 2-ME analogues for biological testing. Lastly, it is
also possible to modify the acetylene coupling reaction shown in
FIG. 1 to prepare 2-(1-propynyl) and 2-(1-butynyl) derivatives of
2-ME that could serve as precursors of 2-propyl and 2-butyl 2-ME
analogues.
[0033] The synthesis reactions in FIGS. 1-4 outlined above will
provide an efficient way of generating 2-ME (analogue 19) and
fourteen 2-ME analogues (analogues 20-33) that can be utilized to
determine the effects of modifying both the C-17 and the C-2
position of 2-ME. Samples of the estrone analogues themselves
(analogues 8-10, 14, 18) will also be tested for their potential
growth-inhibitory activity. The reaction sequences outlined in
FIGS. 1-4 will therefore produce a total of 21 new 2-ME analogues
to be tested as potential selective inhibitors of cancer cell
growth and angiogenesis. It is anticipated that one or more of
these analogues may manifest selective growth-inhibitory activities
towards cancer cells while, at the same time, being less subject to
metabolic conversions that will deactivate or eliminate these
active analogues. It is also likely that 17a-ethylnyl derivative of
2-ME may have a longer effective half-life both in vitro and in
vivo.
[0034] Referring to FIG. 6, eugenol also inhibits the growth of
LNCaP cells significantly. A concentration of approximately 0.75 mM
was necessary to see 50% inhibition of growth of LNCaP cells
whereas a concentration of more than 2 mM was necessary to see
similar effect in DU145 cells.
[0035] The investigational work of the present inventors establish
that eugenol works in combination with 2-ME to achieve even more
impressive results than either substance alone. Cells were treated
with either eugenol (0.25, 0.5, 0.75 or 1 mM) or 2-ME (0.5, 1, 2 or
3 mM) or both (0.25, 0.5, 0.75 or 1 mM of eugenol along with 0.5 mM
of 2-ME). Cell growth was measured following 72 hours of treatment
as described above. As shown in FIG. 7, 0.5 mM of 2-ME inhibited
growth of LNCaP cells by about 20% and 0.25 mM of eugenol inhibited
the growth by about 30%. However, combining both the agents showed
more than 50% inhibition thereby establishing a synergistic
activity of eugenol and 2-ME in combating cancer cells.
[0036] The mechanisms of action at work against the cell lines
investigated thus far are reasonably expected to be equally
efficacious in treating other cancers and pre-cancerous conditions,
such BPH and the cancers of brain, liver, lung, colon and skin, and
in preventing initial onset of cancers and preventing recurrence of
cancers after treatment (such as prostectomies). Since both
hormone-responsive and hormone-refractory prostate cancer cells are
inhibited by 2-ME and its analogs, with or without synergistic
compounds such as eugenol, patients can be treated with these
agents after surgery to prevent the recurrence of
hormone-refractory cancer.
[0037] Additionally, the analogues of 2-ME described above are
expected to provide even greater efficacy, along and in combination
with synergistic, similarly structured compounds as eugenol. This
expectation is well-founded on the efficacy indications established
for 2-ME and the effect of the above-taught structural changes to
2-ME as indicated by the work of the present inventors.
[0038] Application to existing, in vivo tumors may be of varying
means, including, but not limited to, direct injection of the
herein described agents, electrophoresis, and non-electromotive
transdermal migration. Practitioners skilled in the use of
chemopreventative agents will adjust dosages to meet the apparent
needs of any particular patient, and the disclosure contained
herein shall provide an enabling disclosure for the use of 2-ME and
its analogs respectively alone, and with the synergistic compound
of eugenol in the prevention of cancerous tumors as well as the
suppression of recurrent cancers after treatment such as
surgery.
* * * * *